A female infant with Frasier syndrome showing splice site mutation in Wilms' tumor gene (WT1) intron 9.

Wilms' tumor gene (WT1) abnormality leads to various disorders of differentiation as well as renal and urinary system abnormalities. Here we present a case of WT1 abnormality and steroid-resistant nephrotic syndrome in a female infant. The 3-year-old patient was initially diagnosed with proteinuria at an annual mass screening program for children aged three years and was referred to our hospital. She met the diagnostic criteria for nephrotic syndrome and showed normal renal function. The patient was treated with corticosteroids; however her condition showed resistance to corticosteroids. On renal biopsy, she was diagnosed with focal segmental glomerulosclerosis (FSGS). Because of the possibility of WT1 abnormality, an exon array analysis was conducted, which ruled out Denys-Drash Syndrome (DDS). The patient was then diagnosed with Frasier Syndrome (FS) on the basis of donor site mutation (IVS9+5G > A) of the splice site in the intron 9. Reports of female infants with FS are extremely rare. FS is one of the pre-mRNA splicing diseases, in which the occurrence of symptoms is associated with a decrease in the ratio of the lysine-threonine-serine (+/- KTS) isoform of the WT1 protein. A typical case exhibits 46 XY male karyotype and is characterized by male pseudohermaphroditism with cord-like gonadal structures as well as progressive nephropathy caused by FSGS. However, in female infants without such extrarenal signs, it is necessary to consider the analysis for WT1 intron 9 for conclusive diagnosis of FS, because the presence of nephropathy is the only symptom for possible detection.

Partially disturbed lamellar granule secretion in mild congenital ichthyosiform erythroderma with ALOX12B mutations.

Congenital ichthyosiform erythroderma (CIE) (OMIM 242100) is a major type of autosomal recessive congenital ichthyosis (ARCI) showing generalized scaling and erythroderma without blister formation. Mutations in ALOX12B (OMIM 603741), encoding 12R-lipoxygenase (LOX), were identified in patients with CIE in 2002. To date, several ALOX12B mutations have been reported in CIE families. LOXs are a family of nonhaem, iron-containing dioxygenases which catalyse dioxygenation of fatty acids with one or more (Z,Z)-1,4-pentadiene moieties. Three members of the human LOX family, 15-LOX-2, 12R-LOX and eLOX-3, are preferentially expressed in the skin. The 12R-LOX pathway leads to hepoxilin B3 and trioxilin B3 resulting in 20-carboxy-trioxilin A3, which is thought to be a key biological regulator in the skin. 12R-LOX deficiency results in a CIE phenotype in humans and in mice. We report that a Japanese patient with CIE, harbouring one previously unreported ALOX12B mutation p.Arg442Gln and another known mutation p.Arg432X, showed partially disturbed secretion of lamellar granule (LG) contents in the epidermis.

Simultaneous five cell-lineage flow cytometric analysis system for detection of leucocyte antibodies.

Although flow cytometric (FCM) analysis is one of the most widely used approaches to screen the presence of leucocyte antibodies, it has several drawbacks. First, neutrophils and, especially, monocytes exhibit high background reactivity. Second, to determine antibody specificity, it is often necessary to examine not only neutrophils and monocytes but also other lineage cells including T cells, B cells and platelets. Therefore, we attempted to establish an FCM analysis system in which four lineages of leucocytes and platelets are simultaneously tested with low background. FCM analysis was performed using ethylene diamine tetraacetic acid-anticoagulated whole blood as cell sample without any cell preparation. Discrimination of five cell lineages was carried out based on the differences in forward vs. side scatter distribution and in the expression of CD4, CD20 and CD14. When anti-HNA (human neutrophil antigen) 1b antiserum was applied to HNA 1b-positive blood samples, only neutrophils were unambiguously positive. When anti-Naka (anti-CD36) antiserum was applied, only platelets and monocytes were positive. The background reactivity of neutrophils and monocytes was low enough. When anti-human leucocyte antigen (HLA) class II antiserum was tested, only B-lymphocytes and monocytes were positive. When anti-HLA class I antiserum was tested, all the five-lineage cells were positive.

A boy with Japanese Dent's disease exhibiting abnormal calcium metabolism and osseous disorder of the spine: defective megalin expression at the brushborder of renal proximal tubules.

We encountered a 16-year-old boy with Japanese Dent's disease who exhibited renal insufficiency and an osseous disorder of the spine. Proteinuria first was noted at the age of 2 years. At 13 years, the patient underwent analysis of the CLCN5 gene, which identified missense mutation (I524K) in exon 10. During follow-up, a marked increase in urinary beta2-microglobulin was associated with mild deterioration of renal function. At the age of 15 years, hypocalcemia (7.5 mg/dl) accompanied by an increased plasma concentration of alkaline phosphatase was first detected. At that time, plasma concentration of 25(OH)D3 and 1'alpha25(OH)2D3 were low accompanied by a high plasma parathyroid hormone concentration. A renal biopsy specimen revealed tubulointerstitial alterations including mononuclear cell infiltration, partial fibrosis and focal glomerular sclerosis. Immunofluorescence revealed weak, discontinuous staining of megalin along the brushborder of renal proximal tubules. Western blotting demonstrated decreased urinary excretion of megalin. Thus, clinical manifestations and prognosis may vary in Japanese Dent's disease. Reduced megalin expression may have disturbed calcium homeostasis, leading to osseous disorder in our patient.

Cladospolide D, a new 12-membered macrolide antibiotic produced by Cladosporium sp. FT-0012.

A new antibiotic termed cladospolide D was isolated along with the known cladospolides A and B from the fermentation broth of Cladosporium sp. FT-0012 by solvent extraction, ODS column chromatography and preparative HPLC. The structure of cladospolide D was deduced to be (E)-2-dodecen-5-hydroxy-11-olide-4-one. Cladospolide D showed antifungal activity against Pyricularia oryzae and Mucor racemosus.

Deacetylravidomycin M, a new inhibitor of IL-4 signal transduction, produced by Streptomyces sp. WK-6326. II. Structure elucidation.

The structure of deacetylravidomycin M, an inhibitor of interleukin-4 signal transduction, was elucidated to be 6H-benzo[d]naphtho[1,2-b]pyran-6-one, 4-[3,6-dideoxy-3-(dimethylamino)-alpha-altropyranosyl]-1-hydroxy-10,12-dimethoxy-8-methyl- by spectroscopic studies including NMR measurements.

Allopurinol hypersensitivity syndrome associated with systemic cytomegalovirus infection and systemic bacteremia.

A 43-year-old man developed fever, skin rash, eosinophillia, and severe renal and liver dysfunction following treatment with allopurinol. The patient died after 3 months of hospitalization. Autopsy revealed systemic cytomegalovirus infection and bacteremia.

Role of natural killer cells in resistance against friend retrovirus-induced leukemia.

We have previously shown that immunization with a synthetic peptide that contains a single CD4(+) T-cell epitope protects mice against immunosuppressive Friend retrovirus infection. Cells producing infectious Friend virus were rapidly eliminated from the spleens of mice that had been immunized with the single-epitope peptide. However, actual effector mechanisms induced through T-helper-cell responses after Friend virus inoculation were unknown. When cytotoxic effector cells detected in the early phase of Friend retrovirus infection were separated based on their expression of cell surface markers, those lacking CD4 and CD8 but expressing natural killer cell markers were found to constitute the majority of effector cells that lysed Friend virus-induced leukemia cells. Depletion of natural killer cells by injecting anti-asialo-ganglio-N-tetraosylceramide antibody did not affect the number of CD4(+) or CD8(+) T cells in the spleen, virus antigen-specific proliferative responses of CD4(+) T cells, or cytotoxic activity against Friend virus-induced leukemia cells exerted by CD8(+) effector cells. However, the same treatment markedly reduced the killing activity of CD4(-) CD8(-) effector cells and completely abolished the effect of peptide immunization. Although the above enhancement of natural killer cell activity in the early stage of Friend virus infection was also observed in mice given no peptide, these results have demonstrated the importance and requirement of natural killer cells in vaccine-induced resistance against the retroviral infection.

Roles of endogenous retroviruses and platelets in the development of vascular injury in spontaneous mouse models of autoimmune diseases.

MRL/MpJ-lpr/lpr (MRL/lpr) mice spontaneously develop immune complex-mediated glomerulonephritis, granulomatous arteritis, and thrombocytopenia. Recent genetic analyses in a few different strains of lupus-prone mice have pointed out a close correlation between autoantibodies reactive with the endogenous retroviral env gene product, gp70, and the development and severity of glomerulonephritis. We have also shown that autoantibodies reactive with endogenous retroviral gp70 are closely correlated with the development of necrotizing polyarteritis in another lupus-prone strain of mice, SL/Ni. However, suggested pathogenicity of anti-gp70 autoantibodies has not yet been directly tested. To examine if anti-gp70 autoantibodies induce glomerular and vascular pathology, we established from unmanipulated MRL/lpr mice hybridoma clones that secrete monoclonal antibodies reactive with endogenous xenotropic viral env gene products. As reported separately, a high proportion of these anti-gp70 antibody-producing hybridoma clones induced in syngeneic non-autoimmune and severe combined immunodeficiency mice proliferative or wire loop-like glomerular lesions with granular deposits of gp70, IgG, and C3 in affected glomeruli. Some mice transplanted with these anti-gp70 autoantibody-producing hybridoma cells also showed massive subendothelial deposition of electron-dense materials in small arterioles in the kidneys. Furthermore, we identified an IgG2a-producing anti-gp70 hybridoma clone that induced microvascular intraluminal platelet aggregation, thrombocytopenia, and amenia upon transplantation into syngeneic non-autoimmune mice. This anti-gp70 autoantibody bound onto the surfaces of mouse platelets, and specifically precipitated a platelet protein with an approximate relative molecular mass of 40000. Attachment of activated platelets to the intimal surfaces of small arteries was also observed by electron microscopy in mice transplanted with the pathogenic anti-gp70 IgG2a-producing hybridoma cells, suggesting an interaction between antibody-bound platelets and endothelial cells.

Biophysical assessment of persistent effects of moisturizers after their daily applications: evaluation of corneotherapy.

BACKGROUND: Although effective moisturizers can improve xerotic skin changes immediately, their effects are only transient, because the materials applied to the stratum corneum (SC) are easily shed from the skin surface by the daily desquamation process. However, there are a few lines of clinical as well as experimental evidence suggesting that, once application of effective moisturizers is repeated daily, they may produce persistent effects without being influenced by the desquamation of the skin surface. If we can expect such pharmacological effects by simple repeated applications of moisturizers on the skin surface, it will provide a great motivation for the introduction of corneotherapy into the treatment of xerotic skin problems. OBJECTIVE: This study was designed not only to confirm the feasibility of corneotherapy but to propose a practical method to assess such long-lasting effects of moisturizers by using biophysical methods. METHODS: We conducted applications of various moisturizers twice daily to different areas of the flexor surface of the forearms for the initial 5 days of the first week. Thereafter, we performed biophysical measurements of the SC of these areas in the second week, namely 3, 5 and 7 days after their last applications. RESULTS: Daily repeated applications of moisturizers did not induce any change in the water barrier function of the SC or in the size of desquamating corneocytes, a parameter for turnover rate of the SC. However, they substantially increased high-frequency conductance, a parameter for the hydration state of the skin surface, for several days in both normal individuals and patients with atopic xerosis, although the lasting effects were shorter in the latter. The obtained data enabled us to rank the efficacy of moisturizers either according to the duration of the lasting effects or the magnitude of an increase in the hydration levels of the SC. CONCLUSION: The present results confirmed the feasibility of corneotherapy, in which even simple application of moisturizers targeted at the SC can produce unexpected persistent clinical effects after their repeated treatments. The method described in this study constitutes a practical assay system to evaluate the efficacy of topical agents used for dry skin problems objectively and quantitatively.

Comparative study of the efficacy of various moisturizers on the skin of the ASR miniature swine.

The skin of the back of the ASR hairless miniature swine shows a rough and scaly fissured appearance. Clinically it resembles that of ichthyosis vulgaris or senile xerosis in humans. By using the skin of this animal, we developed an assay system to evaluate the efficacy of different moisturizers. The study was carried out in winter months from January to March. To establish this system, we studied various topical agents including such ordinary emollients as petrolatum and 10% salicylic acid in petrolatum as well as those available as pseudodrugs from cosmetic companies or prescription drugs from pharmaceutical companies such as heparinoid- containing creams (w/o and o/w) and 10% urea creams. Applying 0.01 g of each agent to 7 randomly selected skin areas (2x2 cm(2)) on the back of a 6-month-old swine once daily for various periods of time, we measured high-frequency conductance, the parameter for the hydration state of the skin surface. As a result, we have established a simple assay system. It consists of applications of topical agents once daily for 5 days from Monday to Friday in the first week and of subsequent measurement of the skin surface hydration state once every week. Evaluation measurements were performed from day 14 to day 21 depending upon their moisturizing effects. We found that heparinoid-containing creams exerted highly effective moisturizing effects on the dry pig skin that lasted even over 3 weeks after discontinuation of the treatments that were carried out for the first 5 days.

A frozen newborn infant: froth in the air-passage after thawing.

We performed an autopsy on a frozen newborn infant who was found in a freezer at -18 degrees C. After thawing, froth emerged from the nostrils and was present in the trachea. Sometimes froth may be seen in the air-passage in cases of strangulation and drowning. In our case, however, there was neither proof of asphyxia due to strangulation nor drowning. The existence of the froth indicates that the infant was probably in a state of respiratory distress before death. Histologic findings of the lung showed that the infant did not suffer from respiratory disorders such as respiratory distress syndrome. Karyopyknosis and vacuolation of the keratinocytes, shrinkage of the hepatocytes, dilatation of the sinusoid, spaces between heart muscle fibers and deep staining of the nuclei and hemolysis were characteristic in our case. This case shows that froth persists in the internal air-passage for a long time as a result of freezing. Moreover, the froth in the air-passage, along with the findings of the lungs, demonstrates that the newborn infant was born alive.

Establishment of monoclonal anti-retroviral gp70 autoantibodies from MRL/lpr lupus mice and induction of glomerular gp70 deposition and pathology by transfer into non-autoimmune mice.

Several strains of mice, including MRL/MpJ mice homozygous for the Fas mutant lpr gene (MRL/lpr mice), F(1) hybrids of New Zealand Black and New Zealand White mice, and BXSB/MpJ mice carrying a Y-linked autoimmune acceleration gene, spontaneously develop immune complex-mediated glomerulonephritis. The involvement of the envelope glycoprotein gp70 of an endogenous xenotropic virus in the formation of circulating immune complexes and their deposition in the glomerular lesions have been demonstrated, as has the pathogenicity of various antinuclear, antiphospholipid, and rheumatoid factor autoantibodies. In recent genetic linkage studies as well as in a study of cytokine-induced protection against nephritis development, the strongest association of serum levels of gp70-anti-gp70 immune complexes, rather than the levels of antinuclear autoantibodies, with the development and severity of glomerulonephritis has been demonstrated, suggesting a major pathogenic role of anti-gp70 autoantibodies in the lupus-prone mice. However, the pathogenicity of anti-gp70 autoantibodies has not yet been directly tested. To examine if anti-gp70 autoantibodies induce glomerular pathology, we established from unmanipulated MRL/lpr mice hybridoma clones that secrete monoclonal antibodies reactive with endogenous xenotropic viral env gene products. Upon transplantation, a high proportion of these anti-gp70 antibody-producing hybridoma clones induced in syngeneic non-autoimmune and severe combined immunodeficiency mice proliferative or wire loop-like glomerular lesions. Furthermore, deposition of gp70 in glomeruli and pathological changes were observed after intravenous injection of representative clones of purified anti-gp70 immunoglobulin G, demonstrating pathogenicity of at least some anti-gp70 autoantibodies.

Induction of microthrombotic thrombocytopenia in normal mice by transferring a platelet-reactive, monoclonal anti-gp70 autoantibody established from MRL/lpr mice: an autoimmune model of thrombotic thrombocytopenic purpura.

MRL/MpJ-lpr/lpr (MRL/lpr) mice spontaneously develop immune complex-mediated glomerulonephritis and thrombocytopenia. Although the presence of cross-reactive anti-phospholipid antibodies in sera of MRL/lpr mice has been demonstrated, possible relationships between detected autoantibodies and the development of thrombocytopenia have not been elucidated. Recent genetic analyses in a few different strains of lupus-prone mice have pointed out a close correlation between autoantibodies reactive with endogenous retroviral env gene product, gp70, and the development and severity of glomerulonephritis. In the process of establishing possibly nephritogenic anti-gp70 autoantibody-producing hybridoma cells from MRL/lpr mice, we identified an IgG2a-producing anti-gp70 hybridoma clone that induced microvascular intraluminal platelet aggregation, thrombocytopenia, and amenia upon transplantation into syngeneic non-autoimmune mice. This and two other anti-gp70 antibodies bound onto the surface of mouse platelets, and purified IgG2a of the anti-gp70 autoantibody induced glomerular lesions with characteristics of thrombotic thrombocytopenic purpura when injected into non-autoimmune mice. The pathogenic anti-gp70 autoantibody specifically precipitated a platelet protein with an approximate relative molecular mass of 40 000.

Structure elucidation of fungal phenochalasins, novel inhibitors of lipid droplet formation in mouse macrophages.

The structures of phenochalasins A and B were elucidated by spectroscopic studies including various NMR measurements. Phenochalasins A and B have the cytochalasan skeleton of the 21,23-dioxa, 17,22-dione moiety containing unique phenyl and O-methyl phenyl residues at the C-10 position, respectively.

Roselipins, inhibitors of diacylglycerol acyltransferase, produced by Gliocladium roseum KF-1040.

Gliocladium roseum KF-1040, a marine isolate, was found to produce a series of new inhibitors of diacylglycerol acyltransferase (DGAT). Four active compounds, designated roselipins 1A, 1B, 2A and 2B, were isolated from the fermentation broth of the producing strain by solvent extraction, ODS column chromatography and preparative HPLC. The highest production of roselipins was observed when cultured in the medium containing natural sea water. Roselipins inhibit DGAT activity with IC50 values of 15 approximately 22 microM in an enzyme assay system using rat liver microsomes.

Mycobacterium avium infection of the skin associated with lichen scrofulosorum: report of three cases.

We report three Japanese children with Mycobacterium avium infection of the skin who also developed lichen scrofulosorum, a previously undescribed association. They were healthy except for the presence of several noduloulcerative lesions associated with multiple asymptomatic papules on the trunk and extremities. Histology of the ulcerative lesions showed features of mixed-cell granuloma, whereas the papular lesions showed features consistent with lichen scrofulosorum. M. avium was identified by polymerase chain reaction-aided DNA-DNA hybridization analysis in specimens obtained from the noduloulcerative lesions. Both the noduloulcerative and the papular lesions responded well to combination chemotherapy consisting of antituberculous agents and antibiotics.